A technology of inhibiting expression of genes is an important tool for developing a therapeutic agent for treating diseases and verifying a target. As the related art for inhibiting expression of a target gene, technologies of transducing a transgene for the target gene have been disclosed. That is, a method of transducing a transgene in an antisense direction based on a promoter (Sheehy et al., Proc. Natl. Acad. Sci., USA, 85:8805-8808, 1988; Smith et al., Nature, 334:724-726, 1988) and a method of transducing a transgene in a sense direction based on a promoter (Napoli et al., Plant Cell, 2:279-289, 1990; van der Krol et al., Plant Cell, 2:291-299, 1990; U.S. Pat. Nos. 5,034,323; 5,231,020; and 5,283,184) have been disclosed.
Meanwhile, recently, it was reported that a gene inhibition or RNA interference (RNAi) phenomenon after transcription is caused by accumulation of a double-stranded RNA fragment having 20 to 25 base pairs, and the double-stranded oligo RNA is synthesized from an RNA template. The double-stranded oligo RNA fragment is named as “small interfering RNA” (hereinafter, referred to as siRNA). Thereafter, it has been demonstrated that the siRNA is an important factor that inhibits expression of genes in various organisms including mammals (Fire et al., Nature, 391:806-811, 1998; Timmons & Fire, Nature, 395:854, 1998; Kennerdell & Carthew, Cell, 95:1017-1026, 1998; Elbashir et al., Nature, 411:494-497, 2001; WO 99/32619). In addition, it was known that a double-stranded siRNA is converted into a single-stranded RNA by an RNA-induced silencing complex (RISC) and then bind to and inactivate the mRNA (Novina & Sharp, Nature, 430:161-164, 2004). As described above, a technology for inhibiting expression of a gene using the siRNA, which is used to inhibit the expression of a target gene in a target cell and observe changes caused by the inhibition, is advantageously used to identify functions of the target gene in the target cell. Particularly, it is expected that inhibiting functions of a target gene in an infectious virus, cancer cells, or the like, may be usefully applied to develop a therapeutic method of the corresponding disease. As a result of in vitro studies and in vivo studies using experimental animals, it has been reported that expression of a target gene may be inhibited by the siRNA. For example, a method of treating cancer cells by inhibiting expression of Bcl2 protein in the cancer cells using an siRNA has been disclosed in International Patent No. WO 03/070969, and a method of treating cancer cells by inhibiting expression of vascular endothelial growth factor (VEGF) protein causing angiogenesis using an siRNA has been disclosed in WO 04/009769.
In addition, siRNA complementarily bind to the target mRNA to regulate expression of the target gene in a sequence-specific manner, it can be advantageously used in a remarkably enlarged as compared to conventional antibody-based drugs or small molecule drugs (Progress Towards in Vivo Use of siRNAs: MOLECULAR THERAPY: 2006 13(4):664-670).
In spite of excellent effects and various usage ranges of the siRNA, but in order to develop the siRNA as a therapeutic agent, it is required to improve the in vivo stability and intracellular delivery efficiency of siRNA so as to effectively deliver siRNA into its target (Harnessing In Vivo siRNA Delivery for Drug Discovery and Therapeutic Development: Drug Discov. Today: 2006 January; 11(1-2):67-73).
In order to improve in vivo stability of siRNA problems associated with innate immune stimulation problem of the siRNA, research into a technology of modifying some nucleotides or a backbone of the siRNA so as to have resistance against nuclease or using a carrier such as a viral vector, liposome, nanoparticles have been actively conducted.
Delivery system using the viral vector such as adenovirus or retrovirus has high transfection efficiency, but immunogenicity and oncogenicity are also high. On the other hand, a non-viral delivery system containing nanoparticles are evaluated to have low intracellular delivery efficiency compared to a viral delivery system, but has advantages, including high safety in vivo, target-specific delivery, have an improved delivery efficiency due to uptake and internalization of RNAi oligonucleotide contained therein by cells or tissue, and does not almost cause cytotoxicity and immune stimulation, such that currently, the non-viral delivery system has been evaluated as a potential delivery system as compared to the viral delivery system (Nonviral Delivery of Synthetic siRNAs In Vivo, J. Clin. Invest: 2007 Dec. 3; 117(12):3623-632).
Among the non-viral delivery systems, in a method using a nanocarrier, nanoparticles are formed using various polymers such as liposome, a cationic polymer complex, and the like, and then siRNA is supported on these nanoparticles, that is, the nanocarriers to thereby be delivered into cells. In the methods using the nanocarrier, polymeric nanoparticles, polymer micelles, lipoplexes, and the like are mainly used. Among them, the lipoplex is composed of a cationic lipid and interacts with an anionic lipid of endosome in cells to destabilize endosome, thereby serving to deliver the siRNA into the cells.
Further, it was known that the efficiency of the siRNA in vivo can be increased by conjugating a chemical compound or the like to an end region of a passenger (sense) strand of the siRNA to allow the siRNA to have improved pharmacokinetic characteristics (Nature 11; 432(7014): 173-8, 2004). Here, stability of the siRNA is changed depending on properties of the chemical bound to an end of a sense (passenger) or antisense (guide) strand of the siRNA. For example, an siRNA conjugated with a polymer compound such as polyethylene glycol (PEG) interacts with an anionic phosphate group of the siRNA in a presence of a cationic material to form a complex, thereby serving as a carrier having improved siRNA stability (J. Control Release 129(2): 107-16, 2008). Particularly, since micelles made of a polymer complex have a structure spontaneously formed while having significantly small sizes and significantly uniform distribution as compared to microsphere, nanoparticles, or the like, which is another system used as a drug delivery carrier, there are advantages in that quality of a product may be easily managed and reproducibility may be easily secured.
Further, in order to improve intracellular delivery efficiency of the siRNA, a technology for securing stability of the siRNA and implementing efficient cell membrane permeability using an siRNA conjugate obtained by conjugating a hydrophilic material (for example, polyethylene glycol (PEG)), which is a biocompatible polymer, to the siRNA via a simple covalent bond or a linker-mediated covalent bond has been developed (Korean Patent No. 883471). However, even though the siRNA is chemically modified and conjugated to polyethylene glycol (PEG) (that is, siRNA is PEGylated), disadvantages such as low stability in vivo and difficulty in delivering the siRNA into a target organ still remain. In order to overcome these disadvantages, a double-stranded oligo RNA structure in which a hydrophilic material and a hydrophobic material are bound to an oligonucleotide, particularly, a double-stranded oligo RNA such as the siRNA has been developed. The structure forms self-assembled nanoparticles named as “Self-Assembled Micelle Inhibitory RNA (SAMiRNA™)” (see Korean Patent No. 1224828). A SNiRNA™ technology has advantages in that homogenous nanoparticles having a significantly small size as compared to existing delivery technologies may be obtained.
Chronic obstructive pulmonary disease (hereinafter, referred to as ‘COPD’), which is one of the representative pulmonary diseases together with asthma, is different from asthma in that COPD is accompanied by irreversible airway obstruction, and is a respiratory disease characterized by abnormal inflammatory responses in the lung, caused by repetitive infection, inhalation of harmful particles and gases, or smoking, and air flow limitation corresponding thereto, which is not completely reversible and is progressive (Am. J. Respir. Crit. Care Med., 163:1256-1276, 2001). The severity of COPD is emerging around the world. The reason is that in 1990, COPD ranked sixth place among causes of death due to disease, but it is predicted that in 2020, COPD will rank third place, and has become a disease of which an incidence rate is uniquely increased among the top 10 diseases. Further, since COPD is predicted to rank fourth place among causes of disabilities due to diseases, it is expected that social and financial burden due to COPD will rapidly increase (Lancet, 349:1498-1504, 1997). COPD, which is a disease caused by pathological changes in the bronchioles and pulmonary parenchyma due to airway and pulmonary parenchymal inflammation, is characterized by obstructive bronchiolitis and emphysema (destruction of the pulmonary parenchyma). Types of COPD include chronic obstructive bronchitis, chronic bronchiolitis, and emphysema. In the case of COPD, the number of neutrophils is increased, and cytokines such as granulocyte macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, interleukin (IL)-8, and macrophage inflammatory protein (MIP)-2 are secreted. Inflammation occurs in the airway, the muscle wall becomes thick, and mucus secretion is increased, thereby causing bronchial obstruction. When the bronchus is obstructed, the lung sac is expanded and damaged, such that, an exchange ability of oxygen and carbon dioxide is damaged, and occurrence of respiratory failure is increased. Even though 8% of adults over the age of 45 are COPD patients in Korea, medical treatment is biased toward only lung cancer, and as a method of treating COPD according to the related art, a therapeutic agent having an anti-inflammatory effect or a bronchodilatory effect is used. However, essential prevention and treatment for COPD using a gene therapeutic agent is not yet sufficiently developed. A representative example of the therapeutic agents having the anti-inflammatory effect includes glucocorticoid, leukotriene modifiers, theophylline, and the like. However, since the glucocorticoid has potent effects, but is administered by inhalation due to its side effects, and it does not selectively inhibit inflammatory reactions, but inhibit all immune responses and anti-inflammatory responses, in some cases, necessary immune responses may also be inhibited. Since side effects of the leukotriene modifier is small, but there is a limitation in the effect thereof, and when the leukotriene modifier is used alone, it is impossible to regulate asthma. Therefore, in most cases, the leukotriene modifiers are auxiliary used. Theophylline has a problem in that effects are not excellent and there is a risk of side effects. Therefore, the demand for a novel therapeutic agent capable of having excellent effects of preventing and treating COPD and decreasing side effects has been urgently required.
Meanwhile, idiopathic pulmonary fibrosis (hereinafter, referred to as ‘IPF’), which is a kind of fibrosis, is a disease in which chronic inflammatory cells penetrate into a wall of the lung sac (alveola) to cause various changes by making the lung become hard, which causes severe structural changes in the lung tissue, such that lung functions are gradually deteriorated to thereby ultimately result in death. However, an effective treatment thereof does not exist yet, and IPF is generally diagnosed only when symptoms appear, and has extremely bad prognosis since a median survival time is only about three to five years. It is reported that an incidence frequency of IPF is about 3 to 5 per 100,000 people in foreign countries, and it is known that mostly, an incidence rate of IPF is increased over the age of 50, and the incidence rate of IPF in men is 2 times higher than that in women.
The cause of IPF has not been yet clearly identified, and it was merely reported that the incidence of IPF is high in smokers, and anti-depressants, chronic lung inhalation due to gastro esophageal reflux, metal dust, wood dust, solvent inhalation, or the like, is a risk factor related to occurrence of IPF. However, factors having a certain causal factors cannot be found in the majority of patients.
It is known that when IPF is not treated, IPF is continuously worsened, and thus, about 50% or more of patients die within 3 to 5 years. In addition, once a lung is completely hardened by fibrosis as the disease progresses, even when any type of treatment is conducted, a patient does not improve. Therefore, it is predicted that even though treatment is conducted, only when treatment is conducted at an early stage, a possibility for an effect to be exhibited will be increased. As the currently used therapeutic agent, a combination therapy method using steroid and azathioprine or cyclophosphamide, has been known, but it is difficult to say that there are special effects, and attempts of several fibrosis inhibitors in animal experiments and small group of patients failed in proving clear effects. Particularly, there is no other effective therapeutic method in patients with end-stage IPF except for lung transplantation. Therefore, the development of a more efficient therapeutic agent for IPF has been urgently required.
Diseases in which for some reason, tissue or organs are consolidated due to excessive fibrosis of connective tissue are collectively referred to as fibrosis, and all processes of fibrosis are the same as those of scar treatment regardless of a site. It has been almost impossible to completely cure fibrosis symptoms up to now, and a method of treating fibrosis has still been developed and studied. An effective therapeutic agent for fibrosis may also be applied to various diseases accompanied with fibrosis as well as cirrhosis, myelofibrosis, myocardial fibrosis, renal fibrosis, and pulmonary fibrosis, which are representative fibrosis diseases. Therefore, the development of an efficient therapeutic agent for fibrosis has been urgently required.
Meanwhile, it is known that amphiregulin binds to an epithelial growth factor receptor (EGFR) to activate an EGFR pathway and participates in cell proliferation, and the fact that expression of amphiregulin may be inhibited by an amphiregulin-specific siRNA, and the amphiregulin-specific siRNA may have a therapeutic effect for specific type breast cancer has been disclosed (Cancer Res., 2008; 68:225-2265). Further, it was reported that cell penetration in inflammatory breast cancer may be suppressed using a shRNA for amphiregulin (J. Cell Physiol., 2011, 226(10):2691-2701), and when expression of amphiregulin is inhibited using an amphiregulin-specific shRNA, pulmonary artery remodeling is suppressed in mice exposed to cigarette smoke. It was reported that amphiregulin is related to airway smooth muscle (ASM) hyperplasia and angiogenesis, and particularly promotes airway remodeling in asthma patients, and an epidermal growth factor (EGF) excessively secreted in tissue remodeling in acute asthma and amphiregulin are associated with each other.
In addition, it was reported that stratifin (14-3-3 sigma protein or SFN) participates in various intercellular functions such as cell cycle, apoptosis, signaling mechanism regulation, cellular trafficking, cell proliferation and differentiation, cell survival, and the like (Mol. Cell Biochem., 2007, 305:255-64), and participates in TGF-beta 1-mediated growth inhibition using a stratifin-specific siRNA (Mol. Cell, 2010; 0.2; 305-309). In addition, it was reported that a factor regulating formation and decomposition of collagen participates in the airway remodeling in asthma, and particularly, metalloproteinase (MMP)-1 performs an important function in decomposition of collagen, and one of the important factors regulating expression of MMP-1 in the airway is stratifin.
As described above, possibilities of amphiregulin and stratifin as targets for treating respiratory diseases and fibrosis, particularly, COPD and idiopathic pulmonary fibrosis have been suggested, but until now, an siRNA therapeutic agent for amphiregulin and stratifin and a delivery technology of the an siRNA therapeutic agent have not been sufficiently developed. Therefore, the demand for an siRNA therapeutic agent capable of specifically and highly efficiently inhibiting expression of amphiregulin and stratifin, and a delivery technology thereof is significantly high on the market.